3 replies to this topic

[baboon]

Hi,

I am trying to do IHC using an HRP-labeled secondary antibody. To detect the antibody I am using DAB + Nickel chloride as my substrate.
My question pertains to dissolving DAB substrate. I am trying to dissolve DAB in PBT (0.2% BSA, 0.1% triton X-100 in PBS) but it takes forever to dissolve it. Does anyone know a better way of how to  dissolve DAB?

Thx.

[pesji]

baboon, on Aug 19 2009, 11:14 AM, said:

Hi,

I am trying to do IHC using an HRP-labeled secondary antibody. To detect the antibody I am using DAB + Nickel chloride as my substrate.
My question pertains to dissolving DAB substrate. I am trying to dissolve DAB in PBT (0.2% BSA, 0.1% triton X-100 in PBS) but it takes forever to dissolve it. Does anyone know a better way of how to  dissolve DAB?

Thx.

Check the pH of your pBS it has to be 7.3, I would rather dissolve in pBS only

[baboon]

Thanks,
I shall try dissolving in PBS.....

[miraclestrain]

baboon, on Aug 20 2009, 06:14 AM, said:

Hi,

I am trying to do IHC using an HRP-labeled secondary antibody. To detect the antibody I am using DAB + Nickel chloride as my substrate.
My question pertains to dissolving DAB substrate. I am trying to dissolve DAB in PBT (0.2% BSA, 0.1% triton X-100 in PBS) but it takes forever to dissolve it. Does anyone know a better way of how to  dissolve DAB?

Thx.

Hi there,

           I am using the same stuff, but i am using anti mouse HRP, for the DAB I am following the protocol provided by sigma, which is- Crush DAB- (atleast for 1 hour, to make it as a fine powder), try dissolving it in 3-5 ml warm ethanol, finally disolve it in PBS. I would like to discuss more about this, i am from Melbourne and you are?

MS